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http://dx.doi.org/10.4014/jmb.1502.02029

Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene  

Cho, Min Seok (National Academy of Agricultural Science, Rural Development Administration)
Park, Duck Hwan (Department of Applied Biology, College of Agriculture and Life Sciences, Kangwon National University)
Ahn, Tae-Young (Department of Microbiology, Dankook University)
Park, Dong Suk (National Academy of Agricultural Science, Rural Development Administration)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.9, 2015 , pp. 1401-1409 More about this Journal
Abstract
The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 100 fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.
Keywords
Acidovorax avenae subsp. citrulli; bacterial fruit blotch; YD-repeat protein; detection; real-time PCR;
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